Symposium sections:

Pathological morphology of the Human and Animal Diseases

 
Iatrogenic, Environmental and Experimental Pathology

“Herman Jurak” Round Table on Rheumatological Diseases

Ethical forum - Forensic pathology: Proffesion? Science? Politics?

Dental pathology

Slide Seminars

AUTOPSY AS THE UNIQUE DIAGNOSTIC TOOL FOR TUMOUR MULTIPLICITY AND LATENT CANCERS

F. Silvestri, R. Bussani, C. Rizzardi, N. Pavletic.
Institute of Pathological Anatomy, University of Trieste, Trieste, Italy

Most death certificates do not reflect incontrovertible evidence of cancer when compared to evidence established by autopsy, biopsy, or surgical resection of a malignant tumour as an index of cancer epidemiology and biological behavior. Knowing the frequency with which latent and occult cancers may be encountered at autopsy, the difficulty in deciding whether cancer is the cause of, or even related to, death adds further skepticism about the epidemiological value of cancer mortality figures based on death certificate data.
Another problem is the dilemma of cancer in the elderly: approximately 50% of all cancers occur in persons over 65 years of age and close to 60% of all cancer related deaths occur in the same age group. Nevertheless, cancer may be difficult to diagnose in the elderly because of a “geriatric presentation” of the symptoms.
To determine the prevalence of tumour multiplicity (with or without clinical diagnosis) in an autopsy population highly representative of the city situation, we reviewed all necropsy reports performed at the Institute of Pathological Anatomy of the University of Trieste from January 1^st to December 31, 1997 [2141 adult autopsies]. Tumour multiplicity was found in 51 autopsied subjects [(2.4%) 31 males, mean age: 78 yr., 20 females, mean age: 84 yr.]. Forty-six patients presented two malignant neoplasms, while in 4 subjects there were three tumors in various districts. In one case four primary tumors were evident. In more than 60% of cases tumors were diagnosed only at autopsy. Over 50% of the undiagnosed malignant neoplasms were locally invasive tumors, and in 24% of cases distant metastases were found. A high autopsy rate is the only tool for ensuring biological evolution and accuracy of clinical diagnosis of single or multiple malignant neoplasms.

K. Gall-Trošelj
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia

Medullary thyroid carcinoma (MTC) originates from thyroid parafollicular cells and represents 5-10% of all thyroid cancers. Molecular basis of MTC in multiple endocrine neoplasia type 2 (MEN 2) is a germline mutation of the ret proto-oncogene. On the other hand, mutation can be found in approximately 25-30% of sporadic types of tumor. Little is known about other genes involved in initiation and/or progression of those tumors. Point mutations of ret and 12. codon of K-ras were tested after DNA extraction from 5?m tumor tissue section fixed in formalin by PCR/mutation enriched PCR/RFLP analysis in 36 archive samples of MTC; ret point mutation was found in 13%, K-ras codon 12 in 41,65% and ret + K-ras in 16,65%. In 27,8% of samples mutations were absent. Additionally, in 19 samples loss of heterozygosity (LOH) analysis of tumor suppressor genes (p53, nm23-H1, NF-1, BRCA-1) was performed. LOH of p53 and nm23-H1 was not found. In 4 primary tumors and 2 metastasis LOH of BRCA-1 (loci D17S579 and D17S856) was found. The NF-1 loss of heterozygosity was detected in one primary MTC and one metastasis. Further clonality assay, based of methylation of HuMaRa gene locus, was performed to establish the order of genetic changes; this indirect analysis indicated LOH as an early event in C-cell carcinogenesis. Although the occurrence of MTC has been exclusively connected with changes in proto-oncogene ret, the results of this study show that multi-step nature of carcinogenesis of MTC is a rule, rather than exception.

F. Del Piero, E.A. Blomme*, Ž. Grabarević**
University of Pennsylvania, New Bolton Center, Kennett Square, USA *Searle, Chicago, USA, **University of Zagreb, Croatia

All animals develop neoplasms, either common to any species or specie-specific. Describing all animal neoplasms in detail is beyond the scope of our presentation. Therefore this presentation will emphasize frequent neoplasms of production animals, especially horses, cattle, small ruminants, cameloids, and lagomorphs. Other species developing neoplasms relevant to that species, or that serve as models for the study of human neoplasia will also be briefly discussed. Frequent tumors of production animals include equine sarcoid, benign and malignant melanomas, lymphomas, papillomas, equine granulosa cell tumor, squamous cell carcinomas, equine pituitary adenoma of pars intermedia, rabbit uterine adenocarcinoma, equine pedunculated mesenteric lipomas and Marek disease of chickens. Sarcoids are the most frequent equine neoplasms. Lymphomas are proliferations of neoplastic lymphoid cells of T, B, or non-T non-B phenotype able to affect any species at any age, and mimic several other pathological conditions. Several frequently occurring neoplasms have a viral etiology. Many are caused by different retroviruses: bovine and feline lymphomas, avian leukosis, enzootic nasal carcinoma and pulmonary adenomatosis of sheep, feline osteochondromatosis, murine mammary adenocarcinomas, and walleye (Stizostedion spp.) sarcoma. Others are associated to different viruses: equine sarcoid (bovine papillomavirus), papillomas (papillomaviruses), woodchuck (Marmota monax), heron and duck hepatocellular carcinomas (various hepadnavirus), Marek disease of chickens (herpesvirus), fibropapilloma of green see turtles (herpesvirus?), and renal adenocarcinoma of frogs (herpesvirus). Occurrence of some tumors is associated to parasitic infestations: esophageal and gastric sarcomas associated to spiruroid Spirocerca lupi in Canidae, cholangiocarcinoma in Felidae hosting liver flukes within bile ducts, gastric polyps in non-human primates with Nochtia spp., hepatic sarcomas in rodents associated to tapeworm larval stages. In animals, as in humans, genetic predisposition influences development of neoplasms. Some breeds are predisposed to develop certain neoplasms. For instance, gastric adenocarcinomas are observed in Belgian shepherd dogs but are rather rare in other canine breeds. Boxer dogs and other brachiocephalic canine breeds may develop many multiple tumors (e.g. mast cell tumors, gliomas, oral basisquamous cells carcinomas, hematopoietic, vascular, mammary, testicular neoplasms and others). Large canine breeds are more prone to develop osteosarcoma of long bones. Other tumors are hereditary, such as renal adenocarcinomas of German shepherds or melanomas in gray horses. Several exogenous agents are well-known carcinogens in humans and laboratory animals. In animals, neoplasms can also be induced or associated with foreign substances. Examples include osteosarcomas with metal implant in dogs, post-vaccinal sarcomas of cats, asbestos related mesotheliomas in several species, or cyclophosphamide-associated urinary bladder neoplasms in dogs. These exogenous agents may be part of the natural environment of animals. For instance, bracken fern is a well-recognized cause of urinary bladder neoplasm in cattle. Several spontaneous or experimental animal neoplasms are good models for the study of human neoplasia, such as the woodchuck WHV7 virus associated hepatocellular carcinoma. In addition, recent progress in gene transfer technology has permitted the development of large numbers of transgenic and knockout mouse models, which are extremely valuable for the study of cancer and which have tremendously contributed to our present understanding of neoplasia pathogenesis. The incidence of some neoplasms may vary depending on geographic location. This may be related to differences in culture or way of doing things. For instance, mammary, ovarian and testicular tumors are more frequent in countries were gonadectomy of domestic carnivores are not routinely performed. Canine transmissible venereal tumor is more common in parts of the world where dogs are not restricted to confined areas. Or this may be related to differences in exposure to specific carcinogens. Hence, cutaneous squamous cell carcinomas in white cattle and cats will mostly arise in parts of the world with heavy sun exposure. Veterinary pathologists are specialists with appropriate knowledge and training to properly interpret, diagnose and study pathological conditions in animals, including neoplasms. This gives them a unique opportunity to significantly contribute to the identification of new carcinogens or new mechanisms of cancer development. References are available upon request.

G. Stanta, R. Bussani, S. Bonin*, B. Niccolini*, E. Benedetti*, M. Vučić**
Institute of Pathological Anatomy, University of Trieste, Italy
*International Centre Genetic Engineering Biotechnology, Trieste, Italy
**Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Telomere length is maintained by the ribonucleoprotein enzyme telomerase. The RNA component of telomerase (hTR) is widespread, and only the expression of the mRNA encoding the catalytic protein subunit (hTRT) is correlated with telomerase activity.
We have studied the level of expression of hTR and hTRT in four different models of neoplastic and preneoplastic lesions using the RT-PCR method on RNA extracted from paraffin-embedded human tissues after microdissection. The expression at the mRNA level was compared with the enzymatic activity.
Our results suggest that there may be a reciprocal control at the transcriptional level of the expression of hTRT and hTR which in turn is associated with tumour progression.
We test now the possibility to evaluate telomerase expression instead of activity for clinical diagnosis in oncology.

R. Bussani, C. Rizzardi, N. Pavletic, P. Cusati, F. Silvestri
Institute of Pathological Anatomy, University of Trieste, Trieste, Italy

During recent years a growing attention developed for the problem of tumour multiplicity, and some studies have evidenced the frequency of multiple primary tumors correlated with head and neck cancer and, especially, with laryngeal cancer.
In the present research the risk of developing a second bronchopulmonary primary cancer after histological diagnosis of laryngeal cancer has been investigated, making a distinction between supraglottic and glottic locations. The reason for this distinction is that the larynx is essentially composed of two different parts: the upper one (“digestive”) and the lower one (“respiratory”), each with specific embryological, morphological, and functional characteristics.
Frequency of a second primary lung tumour in patients affected by laryngeal cancer has been evaluated on the basis of 128,532 biopsies and 27,753 autopsies carried out during 10 years at the Institute of Pathological Anatomy of the University of Trieste. Among these cases, 432 laryngeal cancers and 44 synchronous or metachronous pulmonary cancers have been detected (7 during life, 37 at autopsy).
The highest risk of developing a lung tumour has been evidenced in patients affected by supraglottic cancer during first 2 years of follow-up (relative risk [RR]: 32.56 for supraglottic patients versus 5.55 for glottic patients). This is particularly true for patients affected by multicentric supraglottic tumors (RR: 62.5).
A significant hyper-frequency of undifferentiated lung cancers also has been noted in supraglottic patients (RR: 45.45 for supraglottic versus 14.28 for glottic patients).
Information provided by autopsy allows a more realistic and detailed outlining of the issue of tumour multiplicity, stressing the importance of strict preventive and follow-up protocols.

Z. Božić, V. Duančić*, O. Kraus**, M. Belicza***
Urologist, private practice, Zagreb, Croatia
*Department of Epidemiology, Medical Centre for Prevention and Rehabilitation of Heart Diseases, Zagreb, Croatia
**Department of Urology, “Sestre milosrdnice” University Hospital, Zagreb, Croatia
***Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Panonian-Balkan endemic uropathy (PBEU) has recently been defined as a single rural household environmental disease of the entire urinary tract due to common living conditions and nourishment, with imprecisely defined, but usually long latency. Seemingly various clinical manifestations, including chronic renal failure due to endemic tubulointerstitial nephropathy, renal cell carcinoma (RCC) and urotheliomas of all localities, observed with significantly higher frequency in the affected population, represent a complex, however, one unique nosological entity which can be found in one single patient. This conclusion is based on results of previous research by Croatian and foreign investigators, as well as our own recent research.
The starting point of our investigation of the endemic occurrence of RCC, as the integral part of the clinical picture of PBEU, was an epidemiological reappraisal of the five essential previously published studies from Croatia and Serbia, covering the period from 1951 to 1990. Our aim was to assess whether RCC occurs with considerably higher frequency in the endemic area or not. Due to insufficient statistical analysis of their results none of the authors of previous reports observed a definite correlation between RCC and endemic nephropathy. In our comparison of RCC frequency in endemic vs. non-endemic areas we used the chi-square test. Statistical analysis of raw data from insufficiently analyzed tables disclosed highly significant frequency of RCC occurrence in the endemic area. Because of omissions in statistical analysis authors of previous studies failed to recognize this phenomenon.
We conclude that RCC, although unrecognized, has occurred with significantly high frequency in endemic areas of Croatia and Serbia since the very beginning of the research on PBEU in 1951 until these days. This conclusion has prompted us to continue our research on epidemiological and clinical characteristics of endemic RCC in the endangered area. This epidemiological reappraisal revealed the extent of one of numerous gaps in comprehension of the complex problem of PBEU.

C. Bianchi, A. Brollo, L. Ramani, T. Bianchi
Laboratory of Pathological Anatomy, Hospital of Monfalcone, Monfalcone, Italy

Aim. To collect data about incidence of malignant mesothelioma in Central and Eastern Europe.
Methods. A short questionnaire was sent to 83 researchers in following countries: Belarous, Bosnia, Bulgaria, Croatia, Czech Republic, Estonia, Georgia, Hungary, Latvia, Lithuania, Macedonia, Moldavia, Poland, Slovakia, Slovenia, Romania, Russia, Ukraine, Yugoslavia. Questions regarded the approximate annual number of mesotheliomas diagnosed in the country, consumption of asbestos, percentage of lung carcinomas recognized as asbestos-related.
Results. Answers were obtained from 12 countries (Table 1).

Table 1: Mesothelioma in Central and Eastern Europe.
 

Country	Population (in millions)	No. of cases per year	Country	Population (in millions)	No. of cases per year
Russia	147,200	n.a.d.	Slovakia	5,400	20
Ukraine	50,660	9	Croatia	4,770	46*
Poland	38,800	120	Lithuania	3,700	12
Romania	22,570	133	Latvia	2,470	8
Belarous	10,360	n.a.d.	Slovenia	1,950	5
Hungary	10,150	78	Estonia	1,470	5

n.a.d. = not available data; * peritoneal mesotheliomas not included

Conclusions. For some major producers and/or consumers of asbestos, data on mesothelioma incidence are not available. In general, and with some notable exceptions, the incidence of mesothelioma appears substantially lower than in Western Europe. A number of factors could explain such difference including: use of different types of asbestos, lower longevity in Central and Eastern Europe, under-diagnosis, etc.. Concerning the last point, co-operative studies involving different European countries would be opportune, in order to assure sufficient homogeneity in the criteria adopted in the pathological diagnosis of malignant mesothelioma.

S. Gajović, T. Malnar, Lj. Kostović-Knežević
Department of Histology and Embryology, School of Medicine, University of Zagreb, Zagreb, Croatia

Nol1 gene encodes proliferation associated nucleolar protein 1.
Nol1 was originally found as a nucleolus marker of various malignant tissues: carcinomas of lung, liver, lymph system, gastrointestinal tract and brain. In the breast carcinoma the level of nucleolar protein 1 was shown to have a prognostic significance. In addition, nucleolar protein 1 is present in the nucleoli of non-malignant, but rapidly proliferating tissues. Nol 1 expression is regulated during cell cycle: protein synthesis starts in middle G1 phase and peaks in S phase of the cell cycle, which suggests its involvement in increased nucleolar activity associated with cell proliferation. Aim of our study was to find if nucleolar protein 1 is involved in embryo development. Mouse carrying insertional mutation within Nol1 gene was produced using the gene trap method. The expression pattern analysis was enabled by inserted lacZ gene transcribed in frame with Nol1 gene. LacZ gene product, ß-galactosidase, was visualized by color changes of its substrate X-gal in blue. The expression pattern of Nol1 gene was studied on 8.5-19-day mouse embryos. During examined stages of development the expression of Nol1 was found in all tissues but not in every cell of embryo. High staining was found in nucleolus, but low staining was present in the nucleus and in the cytoplasm as well. Function of nucleolar protein 1 was studied by analyzing the phenotype of the mice homozygous for gene trap mutation. Using the PCR method we demonstrated that homozygous mice were not present among living offspring and among 11.5-day embryos. Therefore we assumed that homozygous phenotype is embryonic lethal. We intend to analyze homozygous embryos on blastocyst stage, where we expect more data to explain why embryos die. The understanding of Nol1 function during embryo development should open the path to understand its role in tumorigenesis.

D. Jandrić, Z. Petrović, *A.Kurjak, **M.Belicza, M.Ilijaš, D. Košuta
Department of Pathology and Cytology, Sveti Duh Hospital, Zagreb, Croatia
*Department of Obstetrics and Gynecology, University of Zagreb Medical School, Sveti Duh Hospital, Zagreb, Croatia
Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Transvaginal color and pulsed Doppler sonography can provide a superb image of pelvic organs and precise analysis of blood flow characteristic inside the observed vascular network.
Vasodilatation of preexisting blood vessels and growth of newly formed blood vessels is a characteristic of almost all tumor tissues.
Our goal is to determine weather three-dimensional power Doppler can recognize benign from malignant ovarian tumors according to measured resistance index (RI), where low RI refer to malignant lesions opposite to higher RI in benign ovarian tumors.
During the recent period of time a number of benign and malignant ovarian tumors were histologically evaluated for presence of preexisting and newly formed blood vessels and our results are in good correlation with transvaginal color and pulsed Doppler measured resistance index.
References
Kurjak A, Kupešić S, Grgić M, Ilijaš M, Košuta D, Jukić S. Angiogeneza ginekoloških tumora ispitanih obojenim doplerom. Liječ Vjesn 1995; 117: 139-145.
Kurjak A, Kupešić S, Breyer B, Sparac V, Jukić S. The assessment of ovarian tumor angiogenesis: what does three-dimensional power Doppler add? Ultrasound Obstet Gynecol 1998; 12: 136-146.
Kupešić S, Kurjak A. Three dimensional ultrasound and power Doppler in assessment of uterine and ovarian angiogenesis: a prospective study. Croatian Med J 1999; 40: 413-420.
Predanić M, Kurjak A, Šparec V, Kupešić S. Tumor angiogenesis. Gynaecol Perinatol 1998; 7: 161-167.

F. Del Piero, E.A. Blomme*, Ž. Grabarević**
University of Pennsylvania, New Bolton Center, Kennett Square, PA, USA
*Searle, Chicago, USA
**Veterinary Faculty, University of Zagreb, Croatia

Although it has been observed for decades that neoplastic tissues are more vascular than related normal tissues, it is only since Folkman proposed his anti-angiogenesis hypothesis that it is recognized that tumors require a vascular supply for progressive growth. Because of its central role in pathogenesis of neoplasia, tumor angiogenesis is now considered one of the most important factors of tumor biology. Consequently, the tumor vasculature has become an important target in cancer treatment and an area of intensive research. The regulation of tumor-associated angiogenesis is a complex process involving many factors. In last few years, there have been dramatic advances in our understanding of angiogenesis and in knowledge of factors regulating this process. It is now well accepted that controlling angiogenesis is critical to clinically control cancer and other angiogenesis-dependent diseases. The list of known angiogenic stimulators and inhibitors is continuously increasing. There are now many well-characterized pro-angiogenic and anti-angiogenic agents and factors.
Pro-angiogenic agents can be further divided into several categories: growth factors, proteases, trace elements, oncogenes, cytokines, signal transduction molecules, and endogenous inducers. Some of the most potent pro-angiogenic factors include: vascular endothelial growth factor/vascular permeability factor (VEGF/VPF); basic fibroblast growth factor (bFGF); and hepatocyte growth factor/scatter factor (HGF/SF). Other positive regulators of angiogenesis include: angiopoietin-1; angiotropin; angiogenin; epidermalgrowth factor (EGF); granulocyte colony-stimulating factor (G-CSF); interleukin-1 (IL-1); IL-6; IL-8; platelet derived growth factor (PDGF); and tumor necrosis factor-alpha (TNF-alpha). In addition, matrix proteins (such as collagen and fibrin) and cell surface molecules (especially the integrins) are extremely important regulators of the angiogenesis process. Finally, proteolytic enzymes are also considered to be key regulators of angiogenesis. Those include: cathepsins; urokinase-type plasminogen activator; and several matrix metalloproteinases (especially gelatinase A/B, and stromelysis). Angiogenesis can be physiologically suppressed by several recently characterized endogenous inhibitors such as angiopoietin-2, angiostatin, endostatin, interferon-alpha (INF-alpha), platelet factor-4 (PF-4), prolactin (16kD fragment), thrombospondin, the tissue inhibitors of metalloproteins (TIMP-1, TIMP-2 and TIMP-3), and troponin I. Endogenous inhibitors also include caveolin-1 and –2. These molecules are highly expressed in endothelial cells where they mediate action of endogenous and exogenous factors regulating angiogenesis. Hence, several angiogenic growth factors (bFGF, HGF/SF, VEGF) down-regulate expression of caveolin, while several angiogenesis inhibitors (angiostatin, fumagillin, and thalidomide) up-regulate caveolin expression. For certain factors, exact role played in the angiogenesis process is obscure or variable. Therefore, TNF-alpha, transforming growth factor-beta (TGF-beta), or IL-4 are bifunctional modulators - these molecules are either stimulators or inhibitors depending on several factors such as local concentration, location, microenvironment, or presence of other cytokines. In addition to the recent characterization of these pro- and anti-angiogenic agents, research efforts have focused on molecular mechanisms underlying tumor-associated angiogenesis. A good illustration is investigation linking p53 to the angiogenesis process. Inactivating mutations of the p53 gene occurs in more than half of all human cancers and is also frequent in animal tumors. The mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and malignant progression. Exogenous expression of wild type p53 inhibits angiogenesis in vivo resulting in formation of dormant tumors. By inhibiting angiogenesis, p53 indirectly induces apoptosis in vivo but not in vitro and can revert tumors to a dormant phenotype. It has been shown that using fibroblasts, these cells become fully angiogenic in two steps. Firstly, there is loss of both alleles of wild-type p53, which causes a 20-fold drop in secreted thrombospondin and a fourfold increase in secreted VEGF. Secondly, angiogenic activity increases further following activation of the ras oncogene. Thus, there is a step-wise change in the angiogenic phenotype in response to oncogene activation and tumor suppressor gene loss, resulting in decreased secretion of angiogenesis inhibitors and sequential up-regulation of angiogenesis inducers. Search for efficacious anti-angiogenic therapies is currently a major focus of many pharmaceutical companies and academic institutions, and results from clinical trials are very promising. However, more effort will be required to identify selective and potent inhibitors, to appropriately find the optimal combinations between angiosuppressive and cytotoxic therapies, and to design optimal formulations, routes of administration, and dosing schedules. References are available upon request.

P. Juntes, L. Žnideršič, M. Pogačnik
University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia

Aim of our study was to analyze prevalence of tumors appearing in population of dogs coming as patients to one of the biggest small animal clinics in Ljubljana. Most patients came from the city and its surroundings.
Survey was made on patients that came to a small animal clinics of the Veterinary Faculty in Ljubljana during a period of two years. Dogs were clinically examined. In every tumor case a special protocol was made that included following data: breed, sex, age, reproductive status, other diseases and treatments, was a tumor noticed by the owner and how long before the first visit to the clinic. Whenever possible TNM classification (1) and patho-histological determination was also made.
Results: 7689 of 191 different breeds of dogs were examined and tumors were diagnosed in 467 (6,1%) dogs (2,7% males, 3,4% females; 55 different breeds). They were determined histologically in 262 dogs (56,1% tumors). Mongrels prevailed in the population of examined dogs (26,1), while the most common breeds were alsatian (26,1%), poodle (7,3%) and cocker spaniel (7,2 %), labrador retriever, rottweiler, doberman, golden retriever and boxer (all together 14,3%), the rest were dogs of all other breeds. Foxterier was the breed in which tumors were the most often diagnosed (27,5% of examined foxteriers), next were german pointer (19,5%), boxer (14,1%), middle schnauzer (11,3%), newfoundland (11,3%) and dachhund (10,3 %). In comparison to these breeds tumors were less common in mongrels (3,7%) as well as in other breeds. Most common were mammary gland tumors (35,6%), skin tumors (34,9%), then tumors of digestive tract (9,6%), hematopoietic tissues (4,9%), bones and articles (3,8%), male sex organs (3,6%), female sex organs (2,1%) and respiratory tract (1,9%). Other types of tumors were diagnosed sporadically. The average age in which tumor was detected was the highest in tumors of female sex organs (11,3 years) and the lowest in the central nervous system (average 6,8 years), for other tumors average was between 7,6 and 9,4 years.
Conclusions: Mammary gland and skin tumors were diagnosed most often in the examined population of dogs presenting 70,5% of all tumors.
References
1. Owen LN. Geneva: World Health Organisation, 1980.

M. Tišljar, A. Bauer*, Đ. Vranešić*, V. Savić, G. Krivec, B. Šimpraga,
T. Amšel-Zelenika, M. Balenović, V. Herak-Perković**, A. Nemanić
Poultry Center, Croatian Veterinary Institute, Zagreb, Croatia
^*2Department of Pathology, Clinical Hospital Dubrava, Zagreb, Croatia
**"PLIVA" d.d., Zagreb, Croatia

"Meat spots" or "inclusions" are amorphous material found in the oviduct and in the albumen or chalaza of commercial eggs (Helbacka and Swanson, 1958; Bayer et al., 1974; Solomon, 1999). Since eggs with such "inclusions" are usually of a lower quality, economic losses with this defect being great and reports on "meat spots" in literature are scarce, the initial purpose of this study was to find out the cause of a prolonged occurrence of eggs with "meat spots" in a big ISA Brown commercial layer farm. Ten ISA Brown layer hens were sacrificed under chloroform anesthesia, necropsied and the oviduct was taken for histopathological analysis. Hematoxylin-eosin (HE) was used for staining. These very samples of the oviduct as well as a spleen sample were taken for transmissive electron microscopy (TEM) analysis. A sample of 90 eggs was macroscopically examined on presence of the "inclusions" which were then classified according to their size and the location in the albumen as well as to the correlation of their color with the color and the egg shell quality. The "meat spots" were taken for histopathological analysis and stained with hematoxylin-eosin (HE), using Van Gieson method, Mallory with Crosmann? modifications, Berlin blue with Weckleinfalkenberg? modifications, Kongo red and Von Kossa. Same samples were taken for TEM analysis. Fresh parts of liver, spleen, oviduct, ovary and whole eggs were bacteriologically tested for aerobes. Necropsy did not reveal notable differences between hens and the bacteriological examination did not confirm presence of pathogen bacteria. Microscopic finding of degenerated and/or desquamated epithelium as well as the lymphocytic hyperplasia of various degree in oviductal wall muscular layer was positively correlated with the finding of the greatest number and of the largest size of "meat spots" in eggs. Histopathological analysis showed the lobular conformation of the "meat spot" probably of muscular and connective tissue origin. Virus particles were determined using TEM analysis. Light microscopic finding in the oviduct and in "meat spots" confirmed possible correlation between histopathological changes in the oviduct and "meat spots" formation. As the TEM analysis confirmed presence of virus particles in the "meat spot", and this finding was not mentioned in the literature until now, the procedure of virus isolation is in progress.
References:
Relbacka, N. V. L. et al., Poultry Sci. 1958; 37: 869.
Bayer, R. C. et al., Poultry Sci. 1974; 53: 1768.
Solomon, S. E. et al., Proceed. Eur. Symp. Qualit. Egg. Egg. Product. 19-23 Sept. 1999 Bologna Italy 1999; 2: 43.

H. G. Fassbender
Zentrum für Rheuma-Pathologie, Mainz, Germany

It is as difficult as it is essential to find a suitable animal model for studying etiology and pathogenesis of rheumatoid arthritis (RA). The reason for this dilemma is that RA is composed like a mosaic, consisting of three, obviously unrelated, pathogenic components. To be entitled to be judged a true analogue of the human disease, an animal model would accordingly have to incorporate all three components that are:

• the immunological, inflammatory process,
• the proliferative, destructive process, and
• the primary necrotizing process.

H. Brkić, D. Strinović*, M. Kubat*, V. Petrovečki*
School of Dental Medicine University of Zagreb, Zagreb, Croatia
*School of Medicine University of Zagreb, Zagreb, Croatia

This abstract reports results and methods of dental identification of 1000 human remains exhumed from mass graves in Croatia up to July 1998. Personal identification of the victims was performed at the Department of Forensic Medicine and Criminology at the School of Medicine in Zagreb. A forensic odontologist participated in the identification process by carrying out dental identifications. Dental identification based on available dental antemortem data was achieved in 25% of cases. Dental identification based on dental charts was achieved in 35%, on x-rays in 15%, on photographs of teeth in 22%, on interviews in 18%, and on dental supports in 10 % of the cases. Teeth, in combination with anthropological parameters - age, sex and height as well as with other specific characteristics such as tattoos, personal identification cards, clothes, jewelry and DNA - were helpful for identification of 64% of victims, but their significance was not dominant for the identification. Only in 11% of the cases identification was achieved by other relevant means of identification and teeth were not used at all.
References:
Brkić H, et al., Dental identification of war victims from Petrinja in Croatia. Int J Legal Med 1997; 110: 47.
Strinović D, et al., Identification of war victims in Croatia. Med Sci Law 1994; 34: 207.
Brkić H, et al., The incidence of dental identification from mass graves in Croatia. Acta Stomatol Croat 1998; 32: 415.

M. Marcikić, B. Dumenčić, B. Dmitrović
University Hospital Osijek, Department of Pathology and Forensic Medicine, Osijek, Croatia

A brutal killer succeeded to deceive a medical examiner to determine natural, instead of violent cause of death, what allowed him to legally bury victim’s dead body.
The crime would be hidden forever, but owing to murderer’s daughter who decided to uncover homicide it had come to light three years latter. A case like this could instruct doctors and other medical personnel who are in charge as medical examiners to maintain a good practice of visiting and performing post mortem examination of dead bodies before issuing a death certificate.
Any doctor may be called to confirm death and, in some countries, like Croatia, this same doctor may be able to issue a death certificate that states natural causes of death and avoid further investigation if satisfied by the history and surrounding circumstances. Systems of death investigation differ, but in general those deaths which are criminal, suspicious, accidental, suicidal, sudden and unexpected, unexplained or in any way not due to natural causes must be reported to judges and police in Croatia or to specialized agencies in other countries responsible for medicolegal investigation of death.
We suggest that death investigation strategies should include measures that promote continuity and ongoing experience and education in death investigation.

W.L. Ragland
Department of Avian Medicine, University of Georgia, Athens, U.S.A

Immune suppression of poultry is a serious problem that is not easily monitored. Ideally and practically, a test for immune status should be done with samples collected on one trip to a poultry house. Whereas mRNA in whole blood is sensitive to degradation by ribonucleases, rapid dissolution in Catrimox-14, a cationic detergent, will preserve mRNA for two weeks from 4 to 37 C. Thus, IFN mRNA induced by immune challenge can be collected in 4 hr, and safely transported to a lab for analysis. We have developed a competitive nucleic acid hybridization test for mRNA of chicken IFN alpha and gamma that can be done in microtiter plates. DNA probes for the mRNA were produced with plasmids provided by Drs. J. W. Loewenthal (CSIRO, Australia), M. J. Sekellick and P. I. Marcus (U. Conn., USA). Sample RNA from whole blood was allowed to bind to immobilized capture probe and compete with labeled probe added in excess of the capture probe. Signal was inversely proportional with competition by the mRNA. Specific-pathogen-free chickens had no detectable message before immunization with inactivated Newcastle disease virus (NDV). Kinetics of mRNA induction were the same for both IFN, reaching maximum levels in about 6 hr, and returning to baseline in 7 days. Induction was the same from one day to 4 wk of age, indicating that immaturity of immune responses in the very young is a post-transcriptional event. Chickens were injected intra-celomically with 1, 2.5 and 5-mg cyclophosphamide for 3 days before NDV immunization at 2 wk of age, and samples for IFN mRNA were taken 4 hr later. Levels of mRNA were decreased in dose related fashion, and they correlated with antibody responses one week later. This test can be used for chicken immune status evaluation, and for studying molecular events responsible for immune suppression. Supported by a grant from the US Poultry & Egg Association.

K. Kamino, T. Tillmann
Institute of Experimental Pathology, Hannover Medical School, Hannover, Germany

Aim of the study was to know whether a prezygotic X-ray exposure of male mice enlarges a carcinogenic risk in the progeny. Nine-week-old male CBA/J mice were X-ray irradiated on their testicles (1 Gy or 2 Gy) and were mated at 1, 3 and 9 weeks after treatment with untreated virgin 12-week-old females of the same strain. The 1-Gy offspring (155 males and 127 females), 2-Gy offspring (111 males and 95 females) and additional control offspring (119 males and 111 females) were kept for life under standard laboratory conditions. Lungs were investigated on development of adenomas and adenocarcinomas. Significantly increased tumour incidences was not observed in 1-Gy and 2-Gy offspring groups compared with the control. However, with regard to the dignity of tumors, ratios of animals with carcinoma to tumour-bearing animals were slightly increased in both irradiation groups of descendants. Although this phenomenon is not statistically significant, a possibility of enhanced susceptibility to malignant progression of tumors in the offspring through the paternal germ cell X-irradiation should be considered in connection with our previous investigation.
Reference:
K. Kamino et al., Proceedings of The Japanese Society of Pathology 1998; 87: 367. U. Mohr et al., Carcinogenesis 1999; 20: 325.

F. Bulić-Jakuš, M. Vlahović, G. Jurić-Lekić*, D. Ježek*, T.Belovari**, S. Marić***,
D. Šerman
Department of Biology, *Department of Histology and Embryology, Medical Faculty, Zagreb, Croatia
**Department of Histology and Embryology Medical Faculty, ***Department of Biology, Medical Faculty, Osijek, Croatia

An unique in vitro model of early post-implantation rat development, which results in teratoma-like structures, has been established. The importance of this model for investigation of developmental and teratogenic substances in chemically defined media (Bulić-Jakuš et al. ATLA, 1999, 27:925) led to more precise studies of growth potential and differentiation of various tissues after in vitro culture. Embryonic parts of 9,5-day-old Fischer rat embryos were cultivated for one or two weeks at the air-liquid interface in the protein-free medium (Eagle's MEM), with transferrin or serum. Expression of proliferating cell nuclear antigen (PCNA) was detected immunohistochemically by mouse monoclonal antibody and labeled streptavidin-biotin kit (DAKO LSAB 2 Kit, HRP). Transmission electron microscopy on ultrathin sections was done. Some explants, pre-cultivated in above mentioned media, were grafted under the kidney capsule of syngeneic rats. After two weeks, incidences of tissues were compared by chi-square test. Intranuclear PCNA signal was detected in all in vitro cultivated embryos regardless of the kind of medium used for culture (e.g. in the basal layer of epidermis, mesenchyme, cartilage and among the neural cells) showing that they still posses growth potential. By ultrastructural analysis well advanced differentiation was confirmed in e.g. epidermis (typical keratinocytes connected by desmosomes) or neural tissue (neuropil, myelinization). In teratomas in vivo, a greater variety of even more differentiated tissues was found than in teratomas obtained in vitro (e.g. ganglionic cells, bone, striated muscle), but restriction of developmental potential was sometimes discovered in the case of protein-free pre-cultivated embryos. These results should be viewed as a solid basis for more detailed studies of the impact of developmental and teratogenic factors on post-implantation mammalian development in chemically defined culture conditions which have not yet been developed for whole-rat-embryo cultures.

A. Gaspert, A. Boehler*, W. Weder**, R. Speich*, P. Vogt
Institute of Clinical Pathology, Department of Pathology, *Department of Internal Medicine, **Department of Surgery, University Hospital Zürich, Zürich, Switzerland

Transbronchial biopsy after lung transplantation is, at the moment, the gold standard for the diagnosis of acute rejection and cytomegalovirus pneumonia . Risk factors for development of chronic rejection with a histological aspect of bronchiolitis obliterans are repeated episodes of acute rejection and infections. Both can occur in asymptomatic patients and are sometimes only detected by transbronchial lung biopsy. The aim of the present study was to establish the value of surveillance biopsies in asymptomatic patients, in follow up biopsies 1 month after a previous pathological biopsy result and in transbronchial and surgical biopsies performed for clinical indications.
From November 1992 to February 2000, 92 lungs have been transplanted at the University Hospital in Zürich. 462 transbronchial biopsies and 16 surgical biopsies have been prospectively evaluated for acute and chronic rejection, infection and post-transplant lymphoproliferative disorder using conventional (H&E) and special stains (Elastica van Gieson, Alcian blue and PAS, Grocott), immunohistochemistry and in situ hybridization.
Relevant findings such as acute and chronic rejection, cytomegalovirus, bronchiolitis obliterans organizing pneumonia, pneumonia not otherwise specified and lymphocytic bronchiolitis, were detected in 45% of biopsies in symptomatic patients. Acute rejections requiring therapy were diagnosed in 13% of surveillance biopsies in asymptomatic patients. 70% of acute rejections requiring therapy and 33% of biopsies diagnosing cytomegalovirus were found in surveillance biopsies. A post-transplant lymphoproliferative disorder affecting the lung was detected in a transbronchial and a surgical biopsy in one patient as well as in a surgical biopsy of a second patient. 7 of 11 late (> 45 days postoperative) surgical lung biopsies led to new diagnoses such as relevant acute rejection (1), bronchiolitis obliterans (2), post-transplant lymphoproliferative disorder (2), ischemic necrosis (1), hemorrhage in organization (1) and influenced therapy. In comparison 1 of 5 early (? 45 days postoperative) surgical biopsies yielded a new information, in that case pneumonia.
Transbronchial lung biopsy is important for the detection and distinction of asymptomatic acute rejection reaction and infection after lung transplantation. Surgical lung biopsies yield significant new information influencing therapy when performed late after transplantation. Early detection and therapy of these complications might be effective in reducing incidence of bronchiolitis obliterans and increasing survival rate after lung transplantation.

TUMOUR MULTIPLICITY IN A YOUNG PARAPLEGIC MAN WITH CHRONIC RENAL FAILURE AND SYSTEMIC PSORIASIS

R. Bussani, G. Stanta, P. Cusati, C. Rizzardi, N. Pavletic, F. Silvestri
Institute of Pathological Anatomy, University of Trieste, Trieste, Italy

A 42-year-old man presented with severe microcytic anemia [Hb: 8.3 g/dL] and abdominal pain [left hypochondrium].
His medical history was significant for a 13-year history of psoriasis (complicated with psoriasic arthropathy) and a 10-year history of post-traumatic paraplegia. A chronic renal failure was evidenced in last nine months.
A rectocoloscopy showed small ulcero-erosive areas of rectal mucosa, without signs of atypical cells. Gastroscopy did not evidence significant lesions, but only a moderate chronic gastritis.
Further investigations failed to demonstrate a primary tumour.
Ten days before his death, he presented signs of congestive heart failure with polymorphous sustained ventricular tachycardia. The chest X ray revealed an enlarged left ventricle and a mediastinal unspecified mass. He died from an uncontrollable gastric hemorrhage. The autopsy revealed a poorly differentiated pancreatic carcinoma with neuroendocrine cellular aspects extensively metastasized to the liver, and to the mediastinal, mesenteric, perigastric, peripancreatic, and periaortic lymph nodes. Multiple metastases were found in the left ventricular chamber. Interestingly, a large tubulo-villous adenoma with extensive degenerative areas was found in the right colon, near the ileo-cecal valve. We did not find genetic instability by multiple microsatellite analysis.
The patient presented cystic kidneys, with an evident hypoplasia of the left one.

R.S.U. Hiroshi, D. Jakovčević, M.J. da Silva, O.F. Giannotti
Department of Pathology, University Federal Sao Paulo, Sao Paulo, Brasil

Aim of the study is to analyze immunohistochemical expression of hormone receptors (estrogen –P29 and 1D5; progesterone-1A6), Carcinoembryonic Antigen (CEA), PCNA, C-erbB-2 and P53, comparing to special histological types and grading in 31 cases of invasive breast carcinoma.
Formalin fixed, paraffin embedded tumour tissue of these 31 patients, ranging in age from 21 to 75 years, was stained with antibody to P29, 1D5, 1A6, CEA, PCNA, C-erbB-2 and P53 ( 1A6-Novocastra; P29, 1D5, CEA, C-erbB-2, and p53-Dako).
Twenty three cases were classified as invasive ductal breast carcinoma, non-otherwise specified (NOS): the majority of cases 30,4% were grade I, 43,5% were grade II and 26,1% were grade III. The other 8 cases were of special types. Immunohistochemical analysis revealed following results: 1D5 was positive in 56,5% cases NOS, P29 and C-erbB-2 were positive in 91,3 and 82,6% NOS, CEA and PCNA were positive in 100% cases, 1A6 and P53 were absent in 60,8 and 73,9% cases NOS.
In special types: 1D5 and p53 was positive in 50 % cases, 1A6 was negative in 87,5% cases, P29 and C-erbB-2 were positive in 75 and 87,5% cases, CEA was present in all cases & PCNA was strongly positive (>50%) in 62,5% cases.
NOS carcinomas were further analyzed according to the degree of differentiation. 1D5 positivity was detected in 5/7 grade I, 6/10 grade II & 2/6 grade III cases. All the cases of NOS except 2, were positive for P29, and 4 cases were negative for C-erbB-2. 1A6 positivity was seen in 6/7 grade I, 1/10 grade II & 2/6 grade III NOS cases. PCNA was strongly positive (>50%) in 60% cases grade II and weekly positive (<50%) in 71,4% grade I & 83,3% cases grade III. p53 was present in 2/7 grade I, 2/10 grade II, 2/6 grade III cases.
We did not find a significant difference in examined markers between NOS and special histological types. However, p53 was present more often in special types (50%), than in non-special type tumour (26%). Assessment of clinical and pathological impact of these biological markers on IDC requires evaluation in larger studies.
References:
1.Rosen PP. Breast Pathology 1996; pp. 275-320.
2. Tavassoli FA. Pathology of the Breast 1999; pp. 37-74.
3. Lesser ML et al. J Cancer 1981; 48: 299-309.
4. Bloom HJG et al. J Cancer 1957; 2: 359-377.

E. Karahmet, M. Mrkulić, B. Milić, N. Varatanović, M. Podžo, Lj. Babić
Veterinary Faculty, Sarajevo, BiH

Results of investigations of tumors and connective tissue outgrowths on mammary glands in 52 bitches of different age are presented. Greatest frequency of tumors was found in bitches older than 8 years (21 cases – 40,3%).
The most frequent localization of tumors was on pubic, then abdominal complexes and rarely on thoracic ones. Malignant tumors were found in 42 bitches – 80,7% cases, and benign tumors in 10 bitches – 19.3%.

G. Jurić, K. Žarković, D. Chudy*, I. Aurer**
Departments of Neuropathology, *Neurosurgery and **Internal Medicine, Clinical Hospital Center Zagreb, Zagreb, Croatia

Eosinophilic granuloma is one of a triad of lesions encompassing a disease under the generic name of histiocytosis X or Langerhans cell histiocytosis. Etiology remains uncertain and could be related to undefined immunologic disturbance. It is a disease of children and adolescents that very rarely affects adults, representing almost the localized form of a pathological proliferation of histiocytes in bones. The skull is the most common presentation of the disease, and the associated symptoms depend on the location of the lesion. However, multiple lesions of the skull are rare.
A 25-year-old woman was referred to our hospital because of headaches over the period of three years that manifested in parietal region often accompanied by swelling of galeal soft-tissue. X-ray of the head revealed multiple ovoid and rounded well-limited lytic calvarial lesions particularly found in the frontal and parietal bones. The diameter of the lesions ranged from 15 to 70 mm. CT scan confirmed these lytic lesions; a tail of dural enhancement and reactive changes in the overlying galea were not seen. Radionuclide bone scan showed increased uptake at mentioned sites of the skull. No other localizations of lesions were found. A left frontal craniectomy was performed only for resection of one lesion. The dura mater of that site was also removed and was replaced with a dural patch.
Accumulations of histiocytes admixed with eosinophils, lymphocytes, plasma cells, and neutrophils infiltrated the bone, galeal skeletal muscle and fibrous tissue, and also the dura mater. Occasionally patchy zones of hemorrhage and necrosis were present.
Although the natural history of multiple eosinophilic granulomas remain poorly defined, surgical treatment still remains the mainstay of therapy. In addition, different adjuvant treatments are suggested.

N. Ugarković, J. Talan – Hranilović, M. Belicza
Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

The aim of this research is to find out whether the number of glial cells’ tumors has increased after the war in Croatia or not. Data sources are biopsy registers of the Department of Clinical Pathology of Sestre milosrdnice University Hospital. Total number of brain tumors in 1989-90 came to 361. Out of them 11,91% were metastatic, 36,01% - benign and 52,08% - malignant. Number of glial cells’ tumors came to 175, which is 44,48 % of total number. 62,28% of them were malignant (astrocytoma gradus III, ependymoma anaplasticum, glioblastoma, glioma mixtum anaplasticum, oligodendroglioma anaplasticum), and 37,72% (astrocytoma, astrocytoma pilocyticum, ependymoma, glioma mixtum, oligodendroglioma) were benign tumors. During the post- war period total number of all brain tumors comes to 317. 22,08% of them are metastatics, 33,43% are benign and 44,47% are malignant. Out of all these tumors 117 are the glial cells’ tumors which comes to 36,91% of total number. Up to 76,92% of all glial cells’ tumors are malignant and only 23,08% are benign. According to these results comparing the facts on the number of patients we can see that the number of all brain tumor patients decreased during the post-war period and the number of glial cells tumors as well. However, the fact, that there was a large number of patients from the entire territory of former Yugoslavia, and especially those from Bosnia and Herzegovina, which came to this Hospital, should be taken into consideration. Nowadays we only take patients from Croatia. The important fact for both periods is the proportion of benign and malignant tumors. Regarding all brain tumors it came to an increase of metastatic and malignant tumors. We also noticed a significant increase of malignant glial cells' tumors, which could be in relation with possible influence of war’s stress on frequent phenomenon of malignant primary brain tumors.

TABLE 1. Brain tumors 1989-1990 and 1998-1999.
 

YEAR	METASTATIC number (%)	BENIGN number (%)	MALIGNANT number (%)	TOTAL number
1989-90	43 (11,91%)	130 (36,01%)	188 (52,08%)	361
1998-99	70 (22,08%)	106 (33,43%)	141 (44,47%)	317

TABLE 2. Brain glial cells' tumors 1989-1990 and 1998 -1999
 

YEAR	BENIGN number (%)	MALIGNANT number (%)	TOTAL number
1989-90	66 (37,72%)	109 (62,28%)	175
1998-99	27 (23,08%)	90 (76,92%)	117

TABLE 3. Brain glial cells' tumors 1989-1990, 1998 -1999 showed considering the sex and age.
 

		Male number	average	Female number	average	Total number	average
	Benign	41	27,32	25	28,31	66	27,82
1989-90	Malignant	65	44,8	44	40,23	109	43,83
	Total	106	36,06	69	34,27	175	35,11
	Benign	14	38,71	13	41,1	27	40,63
1998-99	Malignant	46	51,42	44	56,23	90	53,34
	Total	60	45,06	57	48,33	117	48,86

M. Atanacković , J. Sopta, S. Popović, Lj. Poleksić*, S. Mirković
Institute of Pathology, School of Medicine, * Institute of Oncology and Radiology, Belgrade, Yugoslavia

Dedifferentiated chondrosarcoma is a primary bone tumor characterized by the presence of low and high grade sarcomatous components. The high-grade component shows histologic features of either malignant fibrous histiocytoma, osteosarcoma, fibrosarcoma, rhabdomyosarcoma etc.
We analyzed two cases with osteolytic lesions in the femur ( 67 year old woman) and humerus (75 years old man). By performing patho-histological analysis of the material gained through biopsy, we observed a presence of malignant spindle cell tumor proliferation (vimentin and CD 68 positive) in both cases. High -grade sarcomas were composed of spindled, fibroblast-like cells in a storiform arrangement and multinucleated tumor cells. We concluded that tumors are malignant fibrous histiocytoma.
After surgical amputation, analysis of the material showed the concomitant presence of typical enchondroma, low-grade chondrosarcoma and malignant fibrous histiocytoma. The greatest part of tumors resemble to enchondromatous proliferation. It was positioned clearly apart from malignant spindle cell tumor component of identical characteristics as seen in the material of biopsy. Chondral tissue showed characteristics of low-grade chondrosarcoma at a very few points and was vimentin and S-100 protein positive.
Radiographic examination (osteolytic lesions with focal calcifications of pop-corn density), SC (pathologic accumulation of radioactive isotope at multiple points) as well as path-histological analysis (enchondroma, low-grade chondrosarcoma, malignant fibrous histiocytoma) made us conclude that, in fact, this were dedifferentiated secondary chondrosarcomas, probably generated on the basis of enchondromatosis, and with low-grade component located in the juxta-position with malignant fibrous histiocytoma.
References:

1.  Ishida T., Kuwada Y., Motoi N. et al :Dedifferentiated chondrosarcoma of the rib with a malignant mesenchymatous component: an autopsy case report, Pathol Int 1997, 47, 397-403
2. Yamaguchi T., Tamai K., Honma K. et al :Malignant fibrous histiocytoma arising from a calcifying enchondroma-a case report, Int Orthop, 1997, 21, 349-51
3. Lee FY., Mankin HJ., Fondren G. et al : Chondrosarcoma of bone: an assessment of outcome, J Bone Joint Surg Am, 1999, 81, 326-38

D. Jelašić, Lj. Banfić*, S. Čužić, M. Čačić, Z. Hutinec
Institute of Pathology, *University Clinic of Cardiovascular Diseases, Clinical Hospital Center Rebro, Zagreb, Croatia

Authors present an unusual case of isolated right heart metastasis of a very rare soft tissue tumor, extraskeletal myxoid chondrosarcoma, its clinical course and treatment in 46 years old female patient.
The patient was accidentally referred to our Hospital because of recurrent pulmonary embolism. Her previous medical history was unremarkable until the age of 40, when a tumor on the right ankle was discovered and surgically removed. Patohistological diagnosis was myxoid chondrosarcoma.
At admittance, echocardiography revealed a large tumor in the right ventricle. Chest CT scan confirmed the large tumor in the right heart, 8 cm in diameter. The most of the right ventricle cavity was occupied with immobile masses that arouse from the right ventricle free wall and invaded a part of the ventricle apex and apical part of the interventricular septum. The heart tumour was surgically removed to a large extent.
Macroscopically the tumor was soft to firm, ovoid, lobulated with distinct fibrous capsule. On the cross section it was lobulated with hemorrhages. Its surface was gelatinous. Microscopic findings presented a distinctive histological picture. The tumor consisted of rounded or slightly elongated cells separated by variable amounts of mucoid material. Cells possessed a small hyperchromatic oval nuclei and narrow rim of eosinophilic cytoplasm. Tumor cells were intracellular PAS positive. Immunohistochemically, tumor cells were vimentin and less intensely S100 protein positive. Histologic diagnosis was extraosseus myxoid chondrosarcoma.
he patient died 11 month after cardiac surgery.
This report revealed extraordinary event in the cardiac pathology mostly because of a very rare type of tumor. There are no published reports of any solitary cardiac metastasis of this tumor type.
Literature:

1. 1. Lucas DR, Fletcher CDM, Adsay NV, Zalupski MM: High-grade extraskeletal myxoid chondrosarcoma: a high-grade epithelioid malignancy. Histopathology 1999; 35:201-8.
2. 2. Graadt van Roggen JF, Hogendoorn PCW, Fletcher CDM: Review. Myxoid tumors of sort tissue. Histopathology 1999;35:291-312.

G. Đorđević, N. Jonjić
Department of Pathology, School of Medicine, University of Rijeka, Rijeka, Croatia

Clinical data clearly indicates a correlation between tumour neovascularization and aggressiveness of tumour growth, and metastatic spread. The vascular endothelial growth factor (VEGF) is known to represent one of the most important angiogenic factors. Present study was planned to demonstrate angiogenesis by average microvessel density (MVD) in various types of renal cell carcinoma (RCC) regarding the nuclear grade and different histologic type, and to investigate changes in VEGF expression. Surgical specimens from 53 RCC were examined for MVD and VEGF by immunohistochemical staining, using mAb to FVIII to visualize endothelial cells, and pAb for VEGF expression.
Microvessel count according to histological type was the highest in mixed and the lowest in papillary type of RCC. Rise of nuclear grade, in the clear cell type, papillary type and oncocytoma was related to lower MVD. In eosinophil type it was quite opposite, while mixed type did not show variation of MVD regarding the nuclear grade. Percentage of positive cells and the staining pattern for VEGF was different between various types of tumour cells. In general, in eosinophil type of RCC, almost all cells showed diffused expression for VEGF, while in clear cell type percentage of positive cells varied from 20% to 100%, with the most of cells showing membrane of annular staining pattern. Furthermore, it could be observed that in clear cell type expression of VEFG becomes slightly more present with higher nulcear grade. In this preliminary study we could not find a statistically significant correlation between VEGF expression and MVD. Moreover, the observation that VEGF expression is slightly higher in clear cell type of RCC with higher nuclear grade is in contrast with findings of MVD. This study needs further investigation in order to claudicate this heterogeneity of VEGF expression in RCC.

I. Glumbić, A. Reljić, H. Čupić, B. Ružić, J. Katušić, B. Krušlin
Ljudevit Jurak Clinical Department of Pathology, *Department of Urology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Bellini duct carcinoma (collecting duct carcinoma) is a highly malignant neoplasm that arises from the collecting duct epithelium of the kidney and represents approximately 1 % of all renal neoplasms. Bellini duct carcinoma generally pursues a more aggressive course than conventional renal cell carcinoma. Metastases to regional lymph nodes, bone, adrenal glands, lung, skin and meninges have been reported.
114 patients underwent surgery at our Hospital due to malignant tumor of the kidney during a two-year period (1998-1999). Patients ranged from 24-82 years of age. There were 38 (33,3%) female and 76 (66,7%) male patients.
Bellini duct carcinoma was diagnosed in three male patients (2,6%) aging 79, 66 and 67. In two patients the main symptom was lumbal pain and hamaturia in one patient. All patients had regional lymphadenopathy and one had cervical lymphadenopathy due to a metastatic tumor. Two patients died 1 month after surgery. Macroscopic examination revealed tumors located predominantly in the renal medulla and one tumor had a partially cystic appearance. Histologic examination showed prominent microcystic-papillary, tubular, solid or cribriform pattern in desmoplastic stroma. Cells were enlarged, with relatively uniform vesicular nuclei, eosinophilic nucleoli and coarse granular chromatin, indistinct cellular margin and moderate eosinophilic granular cytoplasm. Immunohistochemically, positive staining with cytokeratin, EMA and Ulex Europeus agglutinin was observed.

V. Čačinović, D. Lenz
General Hospital Našice, Našice, Croatia

Primary leiomyosarcoma of the epididymis is extremely rare. Fifteen cases of primary leiomyosarcoma of the epididymis have been found in the literature.
We present a case of leiomyosarcoma of the epididymis occurring in a 77-year-old man.
A patient presented with a swelling in the right side of the scrotum 3-month in duration. A right radical orchiectomy with ligation of spermatic cord at the level of the internal spermatic ring was performed.
The tumor measured up to 6 cm in diameter, isolated from testis and firm, lobulated, encapsulated and well delineated. It completely replaced the epididymis.
Microscopy showed that the tumor consisted of interlacing fascicles. Nuclei were generally, elongated with blunt ends. Multinucleated giant cells and mitotic figures were frequent.
Results of histochemical methods (Mallory, Gomory and PAS) were typical for leiomyosarcoma.
Immunohistochemically, Muscle specific actin and vimentin were positive in tumor cells.
References:
Farrell, M. A. and Donnelley, B.J. Malignant smooth muscle tumors of the epididymis. J Urol 1980;124: 151-3.

V. Radotić, D. Jakovljević, N. Ugarković, A. Jakovljević, M. Belicza
“Ljudevit Jurak” Clinical Department of Pathology, “Sestre milosrdnice” University Hospital, Zagreb, Croatia

Aim of this investigation was to evaluate significance of social difficulties and stress, induced by war activities in the region, as risk factors in development of precancerous and cancerous skin lesions.
In this study we analyze and compare incidence, sex distribution and age dependent characteristics in patients with precancerous (keratosis solaris and Bowen’s disease) and cancerous (basal cell carcinoma, squamous cell carcinoma and melanoma) skin lesions. In our department, skin cancer registry was created using pathological and clinical data of patients with skin tumor biopsies during the eleven year period (1989- 1999). We investigated pre-war data (1989- 1990) and post-war data (1997- 1999 ) in Croatia.
There were 714 above mentioned skin tumor biopsies in pre-war period (1989- 1990) (3.43 % of total department biopsies) from which 77 precancerous lesions biopsies (0.37 % of total department biopsies) and 637 cancerous lesions biopsies (3.06 % of total department biopsies). The histologic type distribution in pre-war period (1989- 1990) was for basal cell carcinoma 471 (66 %) biopsies with M/F ratio 1:1; squamous cell carcinoma in 136 (19 %) biopsies with M/F ratio 1.6:1; keratosis solaris in 70 (9.8%) biopsies with M/F ratio 1:1.1; melanoma in 30 (4.2%) biopsies with M/F ratio 1:1.5; and Bowen’s disease in 7 (1%) biopsies with M/F ratio 1.1:1.3. The mean age at the time of diagnosis in pre-war period (1989.- 1990.) was for basal cell carcinoma 66 years, for squamous cell carcinoma 71 years, for keratosis solaris 67 years, for melanoma 60 years and for Bowen’s disease 70 years. In postwar period (1997-1999) there were 1082 biopsies (2.75 % of total department biopsies) from which 175 were precancerous lesions biopsies (0.44 % of total department biopsies) and 914 cancerous lesions biopsies (2.32 % of total department biopsies). The histologic type distribution in post-war period (1997- 1999) was for basal cell carcinoma 711 (65.7%) biopsies with M/F ratio 1:1; keratosis solaris in 168 (15.5%) biopsies with M/F ratio 1.1:1; squamous cell carcinoma in 140 (12 % ) biopsies with M/F ratio 1.4:1; melanoma in 76 (7%) biopsies with M/F ratio 1:1; and Bowen’s disease in 7 (0.7 % ) biopsies with M/F ratio 1.3:1. The mean age at the time of diagnosis in post-war period (1997- 1999) was for basal cell carcinoma 68.5, for keratosis solaris 67 years, for squamous cell carcinoma 70 years, for melanoma 58 years and for Bowen’s disease 72.5 years.
This data shows an increase of melanoma from 4.2 % in pre-war period (1989- 1990) to 7 % in post-war period (1997- 1999), keratosis solaris from 9.8 % to 15.5 % and a decrease of squamous cell carcinoma from 19 % to 12 % in the skin cancer data bases.
References:

1. Green A. et al; J Epidemiol 1999 Dec; 9 (6 Suppl ): S7- 13
2. Zanetti R. et al; Br J Cancer 1996 73: 1440-6
3. Rosso S: et al; Br J Cancer 1996 73: 1447-54

A. K. Kolomiytsev,
Department of Pathology, Rostov-na-Donu Medical University, Rostov-na-Donu, Russia

Problems of chronic gastric and duodenal ulcer pathogenesis are still far from being completely studied. Attention is mainly paid to local factors maintaining the ulcer persistence, especially H. pylori (1,2). Concrete role of the central nervous system is not understood enough. It is well known that in ulceration degenerative and dystrophic processes are manifested in n. vagus trunks but the state of dorsal nuclei is not studied enough.
The work is based on the following hypothesis. The developmental process is determined by a kinetic curve of population growth, which is typical for every cell association. Every multicellular organism is considered as a system that consists of various cellular associations in symbiotic interaction. One of these associations is dominating and determines developmental kinetics of the whole organism. Nervous system is dominating cell association in mammals. The duration of its development is restricted by the lack of capability of neurons to regenerate in adult organisms. The last may determine the duration of life and persistence of various pathologic processes.
Main purpose of this work is to study alterations in dorsal nuclei of n. vagus system and their role in ulceration.
Research was undertaken on autopsy material. This approach was useful for revealing features of alteration in neural centers that had been accumulated within individual life. Morphological and morphometric studies of n. vagus dorsal nuclei, gastric and duodenal mucosa were applied as the most relevant to autopsy specificity. Cross-sections of medulla oblongata were prepared. A calculation of neurons in the right and left dorsal nuclei per field of vision (280x) was undertaken with mean number identification. This method was applied in order to standardize the research. Nuclear and cellular volumes of neurons were estimated additionally.
35 autopsies were studied: in 20 cases chronic gastric or duodenal ulcer was revealed, 15 cases formed control group.
Results revealed a decrease of neurons amount in transversal sections of dorsal nuclei in medulla oblongata which made up 24,7% (mean quantity) in cases of gastric of duodenal ulcer persistence comparing with control cases. Morphometry also revealed decrease of cellular and nuclear volumes comparing with control ones for 14,1 %.
The study shows morphologic evidence of correlation between alterations in the system of n. vagus and chronic gastric and duodenal ulcer persistence.
References:

1. Cho CH., Gastroenterology, 1996; 110: 1244-52.
2. Peura D.A. , American Journal of Gastroenterology, 1997; 92: 8-13.

J. Sopta, M. Atanacković, S. Popović, Z. Stojšić, Lj. Poleksić*, S. Mirković
Institute of Pathology, School of Medicine, *Institute of Oncology and Radiology, Belgrade, Yugoslavia

Pigmented neuroectodermal tumour of infancy, also known as melanotic progonoma and retinal anlage tumour, is a rare neoplasm in the first years of life. The classic location is the maxilla (80%), but has also been reported in mandible, skull and other bones, mediastinum and epididymis. Clinical radiographic and morphologic characteristics of the tumour resemble those of malignant neoplasm. It is a benign neoplasm of neuroectodermal origin with very good prognosis after radical chirurgical removal. Tumour recidivation and metastasis are very rare after radical resection.
Clinical presentation: A 6-month-old infant had a tumor of the upper jaw. Radical surgery was performed. “Melanotic” tumor was diagnosed on the basis of morphologic properties, special histochemical stain for pigment and immunohistochemistry. Small cells, neuroblast-like, showed only positivity for NSE, and large cells showed melanocyte like features (Masson-Fontana, HMB-45, keratin strong positivity, less so for vimentin and NSE, and negativity for Turnbull Blue). Both cell components were negative for S-100 protein.
Difficulty in making differential diagnosis between “melanotic” tumor and a certain number of malignant tumors in bones of children gave this benign tumor a considerable clinical significance. We want to emphasize the great significance of a detailed histologic analysis and immunohistochemistry for correct diagnosis of the disease and prescription of an appropriate treatment.
References:

1. Dashti SR., Cohen ML., Cohen AR.: Role of radical surgery for intracranial melanotic neuroectodermal tumor of infancy: case report, Neurosurgery,1999, 45,174-8
2. Mast BA., Kapadia SB., Yunis E. et al: Subtotal maxillectomy for melanotic neuroectodermal tumor of infancy, Plast Reconstr Surg,1999, 103, 1961-3
3. el Saggan A., Bang G., Olafsson J.: Melanotic neuroectodermal tumor of infancy arising in the maxilla, J Laryngeal Otol, 1998,112, 61-4

R. Bussani, M. Vučić*, C. Rizzardi, N. Pavletic, F. Silvestri
Institute of Pathological Anatomy, University of Trieste, Trieste, Italy
*Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

Type II glycogenosis type 2 (G2) is a lysosomal storage disorder characterized by an inherited recessive autosomal deficiency of ?-glucosidase. Different phenotypes are described depending to the age of onset and the severity of the disease: the infantile form (early onset and late onset) and a juvenile and adult form. At the present therapeutic experiences are prevalently limited to dietary management of patients with juvenile and adult forms. We report a case of a 3-year-old male, first born of non-consanguineous parents affected by the G2 late infantile form. Diagnosis was suspected at the age of 8 months because of muscular hypotonia and asymptomatic cardiomegaly. He was in good clinical conditions until age of 3, when he was admitted to the intensive care department of our hospital because of severe respiratory insufficiency during an episode of pneumonia. He was dystrophic and presented generalized hypotonia. The patient presented elevated and persistent fever apparently not related to septic episodes and not responsive to any antipyretic treatment. Fever was accompanied by a dramatic decrease in CPK levels. Progressively he fell into a comatose state and died. The fatal course of the disease resembles that of malignant hyperthermia.
At post-mortem examination a conspicuous hepatomegaly was evident. Special histostaining [Best’s carminium] evidenced storage cells in the brain (cerebral cortex, cerebral truncum, cerebellum), in skeletal muscles, in the aorta, in the liver, in the heart and in the lungs. A biventricular active lymphocytic myocarditis was found.
To our knowledge, this is the first documented case of a so heavy brain glycogenic involvement, also with severe cortical lesions.

S. Todorov
Department of Pathology, Medical University, Rostov-on-Don, Russia

A study of 27 autopsy cases was designed to assess relationship of congenital aneurysms of cerebral arteries (ACA) with heart and aorta malformations. The material for this study consisted of necropsy specimens obtained from 16 men and 11 women (average age 42). The saccular ACA were solitary in 20 cases and multiple in 7 cases. 85% of the solitary ACA were combined with other malformations such as hypoplasia of one or both posterior communicant arteries, fenestration semilunar aorta valves, hypoplasia of ascendant and abdominal aorta. Histologically, main morphological changes of ACA walls heart valves and aorta revealed disorganization of collagen, reticulin fibers, abseuse Of elastic membranes, myxomatosis, mucoid swelling due to acid storage, neutral mucopolysaccharides. All these findings may account for ACA as a manifestation with hypoplastic and aplastic vascular signs. As we consider, the most important is the collagen deficit of 3 type in these organs which is the basis of congenital pathology of connective tissue

A. Pešut, S. Džebro, S. Gašparov, D. Ljubanović, G. Gašljević, M. Dominis
Merkur Univerity Hospital, Zagreb, Croatia

H. pylori infection is one of the most common chronic bacterial infections throughout the world. Prevalence of H. pylori infection is 61.4 % in healthy population, according to Croatian epidemiological study. H. pylori infection is recognized as a leading factor in etiology of chronic peptic disease, gastritis, ventricular ulcer, adenocarcinoma and MALT B- lymphoma. The way of transmission is still not yet defined. Results of feco-oral as well as other ways of transmission are contradictory. High risk factors of H. pylori infection are: 1.poor socio-economic conditions, 2. overcrowded living. Psychological status i.e. stress as a possible factor which could influence severity of H. pylori induced changes, is still debatable. Therefore, our aim was to compare two groups of H. pylori positive patients: a group of patients living in prison and a group of "free living" patients. Regarding degree of morphological finding, response to therapy and incidence of reinfection. Patients: 82 patients in prison were matched with 82 free patients regarding H. pylori infection, sex and age. Morphological findings were compared with endoscopic findings, response to therapy, duration of infection free period, and onset of H. pylori reinfection.
References:

1. M.F. Dixon et al.: Classification and Grading of Gastritis; Am J Surg Pathol, 1996, (10): 1161
2. D. Forman: HP infection and Gastric Carcinogenesis; European Journal of Gastroenterology & Hepatology, 1992, 4(suppl 2): S31-S35

V. Pešutić-Pisac, A. Punda, B. Vrbičić, J. Bezić, S. Tomić, A. Alujević
KB Split, Department of Pathology, Split

During last ten years we noticed significantly increased incidence of goiter, thyroiditis, adenomas and carcinomas of thyroid, but in the last five years the number of carcinomas is doubled.
In past two years (1998, 1999) 466 thyroid glands were surgically treated for different etiology, and a frozen section was performed for each of them. Carcinomas constituted 20,8% (97) of all cases. The most common type is papillary (73,5%, 75 cases), and follicular (15,5%, 15 cases) carcinoma. Performing the frozen section 54 of them were recognized as malignant, but 43 patient had to wait for definitive diagnosis until permanent (Hemalaun-Eosin) section evaluation. In such cases surgery had to be redone, and we analyzed reason for that.
After the analysis we stated five cases (3 papillary carcinomas, 1 insular and 1 medullary carcinoma) in which the frozen section was wrongly interpreted as benign or the diagnosis was discouragingly delayed.

T. Belovari, F. Bulić-Jakuš*, G. Jurić-Lekić**
Department of Histology and Embryology, Medical Faculty, Osijek
*Department of Biology, **Department of Histology and Embryology, Medical Faculty, Zagreb, Croatia

Early post implantation rat embryos can differentiate to derivatives of the three germ layers in a serum-free culture system in vitro. The degree of differentiation of the neural tissue can be enhanced by subsequent grafting of such embryos pre-cultivated for 7 days with transferrin (Strahinić et al., Period.Biol. 1996; 98:237).
In the present study grafting was done after 14 days in vitro and the effect of two different protein additives (transferrin or albumin) to the serum-free medium was investigated.
Fisher rat embryos (9,5-days-old) were cultivated in Eagle’s Minimum Essential Medium on the metal grid for two weeks and grafted under the kidney capsule for additional two weeks. Routine histological methods and light microscopy of the Hemalaun-eosin, stained serial sections were used to evaluate differentiation.
Differentiation degree of various tissues found in teratoma-like structures was always higher than one observed after culture in vitro. However, the incidence of tissues did not depend upon the quality of the protein supplement and was always higher than in teratomas originating from protein-free cultivated embryos. The only exception was epidermis with its derivatives.
It can be concluded that epidermis is the least sensitive tissue to the otherwise restrictive culture conditions.

K. Žarković, G. Jurić, D. Štimac*, N. Bešenski**, J. Paladino*
Department of Neuropathology, *Department of Neurosurgery, **Department of Radiology, Clinical Hospital Center Zagreb, Zagreb, Croatia

Pachymeningitis in anterior fossa is rare as resistance of dura mater to infection usually limits the inflammatory process to the epidural space. Occasionally, spread of infection occurred within collagen and elastic fibers layers of the dura mater causing pachymeningitis, which may subsequently spread into the subdural space. Anterior fossa infections are usually spreads from frontal, or other paranasal sinus. Mixed infection of Gram-positive and Gram-negative bacterial pathogens is common. Granulomatous bacterial (tuberculosis, syphilis, brucellosis) and non-bacterial (sarcoidosis, autoimmune inflammation or vasculitis) form of pachymeningitis has also been described and may occasionally involve the scull dura. The granulomatous bacterial pachymeningitis of the vertebral column is more often and may extend from pleural, subphrenic, and perinephric inflammation into the spinal epidural space by means of intervertebral foramina. Pachymeningitis in the anterior fossae can be diagnosed using CT or MRI.
A 50-year-old woman was examined by ENT due to headache. CT scans showed a mass within sphenoid sinus. The mass was partially removed by a flexible endoscope. It was concluded that the mass is a very aggressive inflammation. Progressive visual loss on the right eye occurred after 6 months. MRI showed extensive contrast enhancement and thickening of the dura in tuberculum sellae region on T1 WI after contrast media administration. Overlying brain parenchyma was normal except of a moderate diffuse atrophy. Differential diagnosis included pachymeningitis or meningioma. The patient was operated by right subfrontal approach via eyebrow keyhole. The expansive mass was removed. After operation there was no recovery of the visual function. Ten months later due to painful sensation on the right temporal and zygomatic region additional MRI was done. It has shown an en plaque mass, of some neuroradiological characteristics, of the dura in frontobasal right region, above the roof of the right orbit. On admission, she showed blindness on right eye and no other neurological signs. "Reoperation" was carried out. Same approach was applied (subfrontal approach through right sided eyebrow keyhole craniotomy) firm and grayish mass was removed which affected region of right-sided orbital roof, both anterior clinoidal processes, created a thin ring around right optical nerve. The part of tumor mass in the region of cavernous sinus, posterior clinoidal processus and dorsum sellae was left in place. There was no improvement in right eye visual function and without additional neurological worsening.
The biopsy after first neurosurgical treatment yielded two pieces of hard tissue ranged 4 and 5 mm in diameter. HE sections of paraffin embedded tissue showed subacute inflammatory process in one piece of tissue, and meningothelial proliferation with subacute inflammation in the other. Subacute inflammation was composed predominantly of neutrophils, accompanied by few eosinophils, lymphocytes and plasma cells. Inflammatory cells were spread between collagen and elastic layers of dura and around dural blood vessels and nerves. Pathological diagnosis was pachymeningitis and meningioma, while the meningothelial proliferation was much more extensive than usually seen in inflammation. The biopsy after second neurosurgical treatment yielded few pieces of hard tissue ranged 7 mm in diameter. HE sections of paraffin embedded tissue showed chronic inflammatory process composed predominantly of lymphocytes, plasma cells, macrophages and lymphatic follicles.
Extensive contrast enhancement and thickening of the dura in MRI is not obligatory in plaque meningioma, especially when connected with severe infections of paranasal sinuses. Extensive and aggressive pachymeningitis and dura thickening connected with sphenoid sinus inflammation and without epidural or subdural empyema are extremely rare in anterior fossa and in suprasellar region.

B. Šoštarić, Z. Lipej, Ž. Mihaljević
Croatian Veterinary Institute, Zagreb, Croatia

During the summer of 1997 two carcasses (one male and one female) of roe dear were admitted to our Institute with diagnostic purposes, both animals derived from hunting ground in vicinity of Bjelovar. Due to clearly visible numerous cutaneous masses both animals were traced for several weeks and finally shut down by master of the hunt decision. In anamnesis provided by authorities of that particular hunting ground, it was categorically stated that even oldest hunters of the area have not seen similar condition on the roe dear.
On external examination of both carcasses numerous tumor masses varying in size from mans clenched fist to the size of pea grain were noted all over the body, but particularly numerous in inquinal area. The surface of larger masses was ulcerated while the smaller war covered unheard skin. The cut surface revealed a solid mass of white yellowish color with some vasculature and practically without necrosis.
At the middle spring of 1999 a hunting ground of Međimurje was submitted a skin of freshly flayed female roe dear to our Department for further diagnosis due to numerous cutaneous tumors over the skin. Similarly like in previous case this was the first case of such condition noted at that particular hunting ground. However in anamnesis was mentioned that there is still one more severely affected animal at hunting ground.
Microscopic appearance and distribution of tumors on that skin were comparable to those from Bjelovar.
Histologicaly all cases were compatible with cutaneous fibroma of dear, papilomavirus induced and transmissible lesion of dear. The condition is not previously reported in Croatia in scientific literature, even more, all the hunting clubs questioned for previous notification of disease answered negative.
Further investigation is necessary to answer if the condition is introduced in Croatia or existed previously unnoticed.

S. Čužić, M. Šćukanec-Špoljar, B. Jelaković*, D. Kuzmanić*
Department of Pathology, *Department of Internal Medicine, Clinical Hospital Center Zagreb, Zagreb, Croatia

The aim of this study was to elucidate renal distribution of tenascin (TN) in various forms of glomerulonephritis (GN).
Tenascin is a protein of extracellular matrix distributed in renal mesenchyme during fetal development. In adult kidney TN is found in interstitium of renal medulla and mesangial matrix. The exact role of TN is still unknown. It has been speculated that TN may have a mitogenic effect, may induce loosening of mesenchymal cells or be just a part of exoskeletal framework supporting cells. Nevertheless, experiments in TN knockout mice support the idea that TN could have a protective role in development of GN (1).
We examined normal renal tissue and 55 renal biopsies from patients with various forms of GN by immunohistochemistry (APPAP-method) using a monoclonal antibody against TN, clone TN2 (DAKO). In normal renal tissue TN has been found in interstitium of medulla, while cortex interstitium was devoid of TN. TN was found in epithelial cells of proximal tubuli in cortex and in mesangial matrix. In GN tenascin was de novo diffusely expressed in cortical interstitium, at sites of interstitial inflammation, while interstitial fibrosis sometimes revealed TN, sometimes was devoid of TN. Sites of mesangial proliferation were TN positive, just as foci of glomerular sclerosis. TN was detected in fibrocellular crescents and in thickened Bowman’s capsule. In some cases there was also TN positive staining in muscular layer of arteries.
Our results are in concordance with those of other studies (1,2) showing that TN is an important constituent of extracellular matrix in interstitial inflammation and scarring during GN irrespectable of its etiology.
References:

1. Truong LD et al., Nephron 1996; 72:499
2. Truong LD et al., Nephron 1996; 72:579

G. Gašljević, S. Džebro, S. Gašparov, A. Pešut, D. Ljubanović, M. Dominis
"Merkur" University hospital, Zagreb, Croatia

HD is a heterogenous malignant disease. Morphologically and clinically, there are two main types of the disease: NLPHD which is B cell malignant disorder and classic HD in which malignant cell type is still uncertain, but it is also of possible B cell origin. REAL classification recognizes 4 subtypes of HD: NS, MC, LD and LR. ALCL is classified as "T" or "0" NHL which express CD 30 antigen, as well. There are some other morphological and immunohistochemical similarities between ALCL and HD. ALCL can be morphologically confused with HD, most frequently with NS or MC subtypes. We found that FDCs are present in cases of NS and MC in an uneven distribution. However, FDCs are scarce or absent in ALCL cases. Therefore, we tried to use FDCs as one of diagnostic possibilities to differentiate HD from ALCL. Examination was done on paraffin embedded lymph nodes sections of 49 patients- 23 cases of HD ( 4 of NS I , 4 of NS II , 14 of MC , 1 of IF subtype ) and 26 cases of ALCL. For visualization of FDCs, DAKO's FDC MoAb clone CNA.42 was used. The results showed denser and uneven distribution of FDCs in cases of HD, especially of MC subtype, compared to ALCL cases. Our conclusion is that density of FDCs could be used as one of parameters in differentiating HD from ALCL.
References:

1. S.M. Chittal et al.: The interface of Hodgkin's Disease and Anaplastic Large Cell Lymphoma; Cancer Surv, 1997; 30: 87
2. Hodgkin's Disease Expressing Follicular Dendritic Cell Marker CD 21 Without Any Other B Cell Marker; Am J Surg Pathol 1999;23:363

J. Talan-Hranilović, V. Lupret, T. Sajko, M. Kalousek, H. Čupić
Sestre milosrdnice University hospital, Zagreb, Croatia

This report presents a rare type of aggressive prolactinoma with enormous mass of spheroidal amyloid deposits with scarce cellular tumor tissue and without hormonal serological data at the moment of pathological diagnostic procedure.
In pituitary gland and pituitary adenomas different type of amyloid deposits may be found. Incidence of amyloid deposits found in pituitary adenomas is not low, but spheroidal type of amyloid deposits is rarely present. This type of amyloid is called endocrine amyloid, but there are different opinions about its origin, so this problem remains unsolved.
A 28-year-old man, without previous medical history, was admitted due to visual disturbance and headaches, which lasted for about a month. Magnetic resonance imaging (MRI) showed a large lesion in the sellar and suprasellar region with progression toward the sphenoidal sinus. Patient underwent surgery.
Pathohistological analysis of intraoperative and postoperative samples determined an enormous mass of eosinophilic spheroidal material and scanty cellular tissue consisted of polymorphous and hyperchromatic cells. Immunohistochemical staining for prolactin was positive in tumor’s cells and partially positive in spheroidal deposits. Amyloid was proved in those deposits with Congo-red staining. Other immunostaining as CK, Chr-A and Ki-67 was slightly positive in tumor’s cells, while GH and CEA immunostaining was negative.
Prolactin-positive immunostaining of amyloid deposits may question the opinion that amyloid is produced by adenoma ‘s cells.
Prolactinomas are usually tumors of middle aged men. In this case report we present a young man with specific histological findings. Reviewing the literature we came across only 13 published cases, including two personal observations.
References:

1. Voigt C, Saeger W, Gerigk CH, Ludecke DK. Amyloid in pituitary adenomas. Path Res Pract 1988;183:555-7.
2. Lndolt AM, Hetz PHU. Differentiation of two types of amyloid occurring in pituitary adenomas. Path Res Pract 1988; 183:552-4 .
3. Filppi E,Cornaggia M, Riva C, Turolla E. Spheroidal deposits of amyloid in prolactin- secreting pituitary adenomas. Patologica 1992;84:205-14.

M. Krstulja
Department of Pathology, Medical Faculty, Rijeka, Croatia

Morphology (logoz of morjh), spoken as science of forms, in English language coheres both to morphological sciences and to formal expression. Through an Internet search upon “morphology”, through MEDLINE for the 1996 – 1999 period, references were found mostly referring to formal expression. Form, as visualized organization, and pattern, as a repeatable form, supports diagnosis, while relating form to function. Diagnosis and form belong to morphology of life, which is of utmost importance for medical sciences. The scope of this traits is morphology of life at the level of organelles defined as visualized complementary molecular systems, nucleolus taken as example. Life is available as organized and conserved biological structure at any scale, therefore not as a hierarchy of properties expressed by biological forms.

D. Jurela, R. Periz*, M. Belicza*
D. A. M., Zagreb, Croatia
*Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

The computer system “Thanatos 2000” is consisted of 4 modules: record of biopsies, records of cytology, records of the deceased and records of autopsies.
The module of biopsies enables records of general information about the patient, clinical data, histologic diagnosis with description and keeping of subsidiary macroscopic and microscopic pictures. It enables searching by first name, last name, number of biopsy, social security number, date of test, and number of protocol in a hospital or clinic. In enables elaboration of the following reports: description of patients with particular diagnosis, classification by gender and age, report on pictures, the list of frozen section biopsies and the list of biopsies according to the sort of examination.
The module of cytology enables records of general and clinical information about the patient, cytologic diagnoses and record keeping of subsidiary microscopic pictures. It gives possibilities of searching by the name of patient, cytologic number and social security number. It enables elaboration of reports on pictures according to the year and suspectability.
The module of deceased enables records of all general information about the deceased, records of clinical and pathological diagnoses along with differentiation of the cause of death, direct cause of death and major illness. This module enables the printout of Registration of death and Death certificate forms which should be issued obligatorily for each deceased person for the needs of competent state institutions. It enables searching according to the first name and the last name of the deceased, social security number, pathological protocol or hospital’s protocol. It enables elaboration of reports on the number of deceased in each clinic inside the hospital, and the number of demurrages for deceased.
The module of autopsies enables record of established information by the autopsy of each deceased person. Apart from the descriptive part, there is also the coded part which is divided into 5 groups and serves to analyze the entered data. This module enables record keeping of clinical and pathological diagnoses along with differentiation of major illness, cause of death and direct cause of death as well as macroscopic and microscopic pictures connected to the particular autopsy. It enable elaboration of reports on cause of death and diagnoses.
The database contains one base of diagnoses (10000 records) which is used by all mentioned modules, and according to circumstances, in each module and in each particular case, subsidiary attributes can be added to each diagnosis (clinical, pathological, cause of death, major illness etc.)
It is obvious that our computer system has many advantages and such system should be recommended for use in similar and other departments.

S. Prašović, E. Šatrović, H. Beširović
Veterinary Faculty Sarajevo, Department of Pathology, Sarajevo, BiH

In this review authors discuss a case of malignant mesothelioma in cattle. Considering relevant problems of carcinogen effects of asbestos, and the fact that it is the cause in more than 70% cases of this malignant disease, the aim of this review is to attract attention of our colleagues, and give them basic information (characteristics) about this disease. Because mesothelioma looks like a TB process on the serosa, the problem is differential diagnosis between them. On the other side, the pathogenesis of mesothelioma is not fully studied, in spite of its significance in both veterinary and human medicine. Asbestos was used as a construction material between 70s and 80s, and its pathologic effects we expected to be much more widespread and serious between years 2000 and 2010.

S. Atalay Vural, G. Alçigir, Ş. Berkin
University of Ankara, Faculty of Veterinary Medicine, Ankara, Turkey

Rabies is a lethal viral disease caused nonpurulent encephalitis in human and animals. In histopathologic diagnosis of street rabies virus infection Negri bodies are usually required to confirm the diagnosis of rabies. However, these eosinophilic cytompasmic inclusion bodies can be demonstrated in approximately 74-90% of naturally infected animals and in only 50-80% of tissue specimens found positive by virus isolation methods. On the other hand, these rates are reported as 87-98% in immunoperoxidase and 87-100% in immunofluorescent methods.
The diagnosis of rabies infection in animals inoculated with fixed virus can not be made by histopathological examination based mainly on identifying Negri bodies because the fact that fixed rabies virus cannot identify Negri bodies within the neurons of infected brain. Therefore, in experimental fixed rabies virus infection studies for diagnosis, the screening of rabies virus antigen in affected tissues by using immunoperoxidase an immunofluorescence methods as that of the street rabies infection, is more suitable.
This study was carried out to determine the pathologic changes as well as the localization of the viral antigens in the tissues of 10 goats infected with fixed rabies virus for vaccine production.
Tissue samples of goats infected with fixed rabies virus via intracerebral were taken from central nervous system (Gasserian ganglion, retina and other visceral organs and tissues). These samples were fixed in buffered formalin solution and blocked in paraffin. They were stained with hematoxylin-eosin (H&E) immunoperoxidase (avidin-biotin Complex) and indirect immunofluorescence methods using rabbit antirabies hyperimmune sera*.
Histopathologically, nonspecific inflammatory reactions, degenerative and necrotic changes were detected in sections of the central nervous system in all cases and Gasserian ganglion in 4 cases. Very active proliferations of the lymphoblasts were seen in germinal centers of retropharyngeal lymph nodes in 2 cases. In addition, morphologic changes related to membranous glomerulonephritis were seen in kidneys of other 3 cases. The changes of retropharyngeal lymph nodes and kidneys were evaluated as the vaccine’s reaction resulted from the deposition of antigen-antibody complexes.
In immunoperoxidase method, extra and intra cellular rabies virus antigens were seen at sections of central nervous system in 8 cases, in Gasserian ganglion and retina in 4 cases and retropharyngeal lymph node in 1 case.
In immunofluorescence method, viral antigens in similar localization were also seen at sections of the central nervous system in 9 cases, in Gasserian ganglion and retina in 3 cases. But they were more prominent than seen immunoperoxidase method. In immunofluorescence method, the viral antigen weren’t observed in retropharyngeal lymph nodes.
In both immunohistochemical methods, rabies viral antigens were observed intensively as small and/or big granular of diffuse staining characteristics. Even so, immunofluorescence method gave the better results on the quality and quantity of staining traits of antigens compared to immunoperoxidase method.

* Provided by the Centre of Expertise for Rabies Agriculture Canada Animal Disease Research Institute Nepean, Ontario.

J. Dumančić, Z. Kaić, H. Brkić, M. Dumić*
School of Dental Medicine, University of Zagreb
*University Hospital Zagreb - Rebro, Zagreb, Croatia

Premolar root forms were investigated in a sample of 40 females with Turner syndrome and 39 males with Klinefelter syndrome. Healthy females and males, 80 in each group, served as controls. The aim was to investigate the role of the X chromosome in dental root development.
The presence of various root forms of the upper and lower premolars was noted from the orthopanthomograms. For the Turner sample, standard intraoral radiographs were also analyzed. Premolars were classified according to the method proposed by Midtbo (1), partially modified for the lower premolars. There were three types of the maxillary premolars: 1) one-rooted; 2) two-rooted (separated buccal and palatal roots); 3) three-rooted (two buccal and one palatal root). The mandibular premolars were classified into five groups: 1A) one-rooted with one root canal; 1B) one-rooted with two separate root canals; 2A) two-rooted with separate buccal and lingual root components; 2B) two-rooted with separate mesial and distal root components; 3) three-rooted with two buccal and one lingual root.
The maxillary and mandibular premolars with two or three root components were found significantly more often in the Turner group than in all other investigated groups (p<0,000001). In maxilla 2,9% of P1 were one-rooted, 88,2% two-rooted and 8,8% three-rooted; 28,6% of P2 were one-rooted, 65,3% two-rooted and 6,1% three-rooted. In mandible 21,6% of P1 were one-rooted and 78,4% two-rooted; 51,4% of P2 were one-rooted, 47,3% two-rooted and 1,4% three-rooted. In the lower two-rooted premolars type 2A was 2 – 4 times more frequent than type 2B. Asymmetry in the root number was significantly more frequent only for the lower premolars (p<0,0005).
There was no significant difference between the Klinefelter group and male controls, or between female and male controls.
The findings indicate that premolar root formation is influenced by the X chromosome deficiency. On the contrary, we did not find any evidence of the influence of the supernumerary X chromosome on premolar root morphology.
References:

1. Midtbo M et al., Acta Odontol Scand 1994; 52: 303.
2. Varrela J. Archs Oral Biol 1990; 35: 109.
3. Kaić Z. 7^th International Congress of Human Genetics 1986, Abs No C I.49.

D. Ježek, I. Krhen*, Lj. Banek, I. Rajčan, Z. Mareković*
Institute of Histology and Embryology, Medical School University of Zagreb
*Urology Clinic, Medical Faculty University of Zagreb, Zagreb, Croatia

A 36-year patient was admitted into the hospital because of the nodular changes of the left testis. On gross examination (after orchiectomy) the tumour tissue was predominantly soft and yellowish, with areas of haemorrhagia. A part of the testis that seemed free from the tumour tissue was subjected to the evaluation of spermatogenesis. Histologically, the mentioned part of the specimen consisted of seminiferous tubules with a rather irregular form (folded "lamina propria") and reduced tubular diameter. Within such tubules, a mixed population of tumour cells could be noted. The vast majority of cells looked like spermatocytes/early spermatids with a round, mostly homogenous nucleus and eosinophilic or neutral stained cytoplasm. A minor population of tumour cells consisted of giant cells with oval or irregular nucleus and pale cytoplasm. These cells could be noted not only within the tubules but also in the interstitial part of the gonad. Some giant cells invaded blood and lymph vessels. Few of analyzed seminiferous tubules were fibrosed and transformed into "tubular shadows". Despite of the significant presence of tumour cells, spermatogenesis was maintained within the majority of the analyzed tubules. Spermatogonia, spermatocytes, round and elongated spermatids were observed in the tubules, sometimes mixed with tumour cells. Interstitial Leydig cells were reduced in number and size. Areas of interstitial fibrosis could be also observed. It is concluded that the tumour corresponds to the picture of spermatocytic seminoma, with a characteristic "mixed" population of tumour cells. In the area that was not totally consumed by the tumour, seminiferous tubules still have all types of spermatogenic cells, including round and elongated spermatids. The existence of spermatogenic cells could be explained by the nature of the tumour that is well differentiated and "mimics" normal spermatogenesis.

G. Mazzullo, A. Germaná*, G. Modica**, G. De Vico, G. Germaná*
Department of Veterinary Pathology,
*Institute of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Messina, Messina, Italy
**Veterinary National Health System, ASL 4, Enna, Italy

Conjoined twinning has been reported in most of domestic animals’ species. It occurs extremely rarely in horses, occasionally dogs and cats, and not uncommonly in cattle, pigs and sheep (2). Furthermore, the last species have the highest incidence of craniofacial defects among domestic animals and man (1).
In order to contribute to the knowledge of animal congenital defects, the presented paper describes the morphological features of a diprosopus lamb.
A live male crossbreed dystocic two-headed lamb was delivered from a 2-year-old Pinzerita sheep after first mating. After 40 hours of life, the lamb spontaneously died. It was frozen and then sent to the Department of Veterinary Pathology of the University of Messina for necropsy. Samples of different tissues and organs were collected and processed for routine histopathology.
The lamb had two heads, four eyes and two ears. Skulls were joined in the frontal-parietal-temporal region, partially involving the parotidean and maxillary regions. Medial mandibular branches were joined in the caudal third, nearby the vertical mandible branch. Each head possessed a nasopharynx, an oropharynx and a normal tongue. Soft and hard palates were complete. Oropharyngeal regions of each head merged to form a single laryngopharynx and a single oesophageus. The lamb had two separate brains and cerebella and fusion occurred in the region of the medulla oblongata. The remainder of body tissues and organs were within apparently normal limits.
First examination of different organ and tissue sections did not reveal remarkable histomorphological changes.
The lamb was classified as a conjoined twinning and, on the basis of the facial duplication, as a diprosopus tetraophthalmus.
In conclusion, the authors discuss about the need for further studies in this branch of comparative pathology, which are infrequently reported in the veterinary practice.
References:

1. Dennis SM et al., The Vet Bull, 1979, 49:233.
2. Fisher KRS et al., The Anat Rec, 1986, 214:432.

I. Jurak, K. Gall-Trošelj, J. Pavelić, Š. Križanac*, M. Mrčela**, K. Pavelić
Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia
*Dubrava University Hospital, Zagreb, Croatia
**Osijek University Hospital, Osijek, Croatia

DNA analysis at polymorphic loci 1p31 (D1S500, D1S465, D1S430, D1S207, D1S208, D1S488) and 1p13 (D1S189, D1S252, D1S440, D1S453, D1S514), based on LOH, was performed in one mixed ovarian tumor consisted of yolk sac, dysgerminoma and embryonal carcinoma. Microdissection was performed in order to separate and analyze two components (dysgerminoma and yolk sac tumor). Embryonal carcinoma was not analyzed. The same kind of analysis was performed at BRCA-1 loci (D17S579, D17S856, and D17S855). Informativity of analyzed regions was high, but loss of heterozygosity was not found. These results exclude a possible involvement of 1p31, 1p13 and BRCA-1 in carcinogenesis of this rare type of tumor.

M. Škarica, L. Tuzović, Ž. Bedić-Fegeš, M. Belicza
Ljudevit Jurak Clinical Department of Pathology, Sestre milosrdnice University Hospital, Zagreb, Croatia

This study was undertaken to examine anatomical distribution of colorectal cancer, anatomical extent of disease (stage), age of presentation for different subsite locations, and whether is there any intersex difference in frequency of the disease in our Registry patient population during two years period (1998-1999).
We analyzed pathological, clinical and demographic data found in our Clinical Department of Pathology Colorectal Cancer Registry. The registry was filled with data from pre-surgery and post-surgery biopsy findings in the University Hospital and clinical data both preoperative and postoperative.
Colorectal cancer registry contained 540 patients with diagnosis of colorectal carcinoma. Overall female to male (F/M) ratio was 38.5%: 61.5%. The most common localization with 379 (70.2%) cases was rectosigmoid with F/M ratio 1:1.7. The next localization was descending colon with 81 (15%) cases and F/M ratio 1:1.6. In cecum and ascending colon we found 64 (11.8%) cases with F/M of 1:1. Transversal colon formed the smallest group with 16 (3%) cases and F/M ratio 1:3. There was no difference in anatomical distribution between males and females. The mean age in males was 67.3 years and in females 68.1 years. In females proximal lesions tend to present at later stage than distal, 73 years for ascending colon and 67.3 years for rectosigmoid region. This difference was not significant in males. We also analyzed TNM diagnosis in 326 patients. The most common stage was T3 with 203 (62.3%) cases. The next stage was T2 with 76 (23.3%) cases. T1 was represented in 23 (7%) cases and T4 in 24 (7.4%) cases. There was no intersex difference. The most common N stage was N0 with 166 (51%) cases, N1 was presented in 96 (29.4%), N2 in 63 (19.3%), N3 in 1 (0.3%) cases, respectively. Distant metastases were found in 14 (4.3%) cases.
Sex distribution in our registry is as quoted in literature. Other parameters we analyzed (stage and age of presentation) also follow data quoted in literature. Significant difference is found in anatomical distribution of the disease. Rectosigmoid presentation of disease (70.2%) which is 2 times more common in our population. Cecum and ascending colon takes another 11.8%, which is related to data in literature 1:3.2. Transversal colon (3%) has the greatest ratio to quoted data – 1:6. Descending colon takes 15% and related to literature is 1.875:1.
References

1. Cotran RS, Kumar V, Collins T. Robbins pathologic basis of disease. 6^th ed. Philadelphia: WB Saunders 1999.
2. Chattar-Cora D, Onime GD, Coppa GF, Valentine IS, Rivera L. J Natl Med Assoc 1998; 90:19-24.
3. Semmens JB, Platell C, Threlfall TJ, Holman CD. Aust N ZJ Surg 2000;70:11-8.

S. H. Bambir, S. Helgason, F. Marino*, B. Macrí*
Institute for Experimental Pathology, University of Iceland, Reykjavík, Iceland
*Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Messina, Italy

Tumors in fish are common like in higher animals and humans. They have been detected in all fish taxa, from Cyclostomata to cartilaginous and bony fish, and in almost all organs. Certain tumors are characteristic for some fish species, and their prevalence can increase like an epidemic. Some tumors are very rare, like liposarcoma and rhabdomyosarcoma.
Here we describe different types of tumors observed in fish that we feel are of interest:
A schwannoma, mixed with a fibroma tumour, was diagnosed in a goldfish (Carassius auratus) sent for examination from a private owner. It formed a single nodule that was located on the side above the lateral line, in front of the dorsal fin and penetrating deep into the skeletal musculature.
A single rhabdomyosarcoma tumour was diagnosed from each of two, out of a total number of 238, Atlantic herring (Clupea harengus harengus) that were examined from a commercial catch south off Iceland. In each fish there was a single tumour in the skeletal muscle above the lateral line, anterior to the dorsal fin.
A liposarcoma was detected in the caudal part of two halibut (Hippoglossus Hippoglossus) juveniles from an experimental rearing fish farm. No external symptoms were seen.
A fibroma was diagnosed in three gold fishes sent for histopathological examination from a public aquarium in the city of Tormina (Italy). Fishes showed one or more nodules in different body regions and in one fish tumoral tissue was invading the eye.
A dark red, round shaped, soft mass was detected in the mouth cavity of a fish (Aulopus filamentosus), behind the tongue. The diagnosis was of thyroid carcinoma.
A large fibrosarcoma was detected on the left side of the head of an adult wild fish (Spondyliosoma cantharus) fished by a scuba diver in one of the most important industrial areas in southern Italy. Toxicological exams revealed high heavy metals levels in tissues.
The histopathology of these tumors will be described.
Schwannomas are thought to be the most frequent types of tumors in goldfish. On the other hand the authors know of only one another described case of rhabdomyosarcoma in fish (Oncorhynchus nerca) and to the best of our knowledge, a liposarcoma has not before been described in fish.
Detection and description of fish tumors can be of use in comparative oncology, and provides information on the possible role of genetics, environment and pathogens on tumour formation.

T. Švara, P. Juntes, M. Pogačnik, I. Zdovc, V. Bole-Hribovšek
University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia

Gram negative bacteria Aeromonas hydrophila usually causes septicemia in cold-blooded animal, mostly fish, amphibians and reptiles. Infections with this organism in mammals are not common. Several cases were described so far, mostly in humans and only few in dogs (1,2). Gross pathology and pathohistological lesions found in two cases of puppies that died due to Aeromonas hydrophila septicemia are described.
In the litter of eight doberman puppies all died in the period of 2 to 10 days after birth. Necropsy was made on two of them. Organ samples were collected for pathohistology and bacteriology, and also the imprints of the pleura were made. Tissue sections were stained with hematoxylin-eosin, some of them also with the Gram staining for tissues (Goodpasture) and Sudan stain for fat. Imprints were stained with Giemsa. Culture was made on the blood agar, Drigalski agar and Saboraud Dextrose agar.
Gross pathology and histopathology findings were similar in both puppies. The most evident were diffuse condensation of pulmonary tissue with multifocal areas of necrosis (acute necrotizing bronchopneumonia), Sudan positive droplets in alveoli and bronchioli (aspiration of the milk), serofibrinous pleuritis, enlarged spleen, congestive liver and kidneys, severe desquamative enterotyphlocolitis and multifocal hemorrhages in or under leptomeninges. Several types of bacteria were found in tissues with Goodpasture method: numerous Gram negative rods dominated but there were also many Gram positive cocci and rods. With bacteriological examination Aeromonas hydrophila was isolated from the lung, liver, spleen, kidneys and intestines - in the later non-hemolytic E.coli was also isolated. The source of infection in puppies was not determined.
References:

1. Ghenghesh KS et al.. Comp Immunol Microbiol Infect Dis 1995; 22: 175
2. Andre-Fontaine G et al. Comp Immunol Microbiol Infect Dis 1995; 18: 69

Z. Šimec, Ž. Grabarević, B. Artuković, S. Seiwerth, Z. Janicki, B. Krušlin
Faculty of Veterinary Medicine and School of Medicine of the University of Zagreb; Zagreb, Croatia

A case of hemangioma in wild boar was described. The tumor was histopathologically and immunohistochemically characterized. Immunohistochemical detection’s of von Willebrand’s factor (vWF), CD31, CD34, Ulex Europaeus agglutinin 1 (UEA), vimentin and keratin from formalin fixed paraffin embedded tissue sections were performed. Gross and histological appearance of the tumor closely resembled to those previously described in domestic pig. Immunohistochemically, anti CD-34 and vimentin were noted to be most sensitive markers in both cavernous and capillary areas of tumor, whereas UEA was observed only focally. In this case the keratin staining was negative. It could be concluded that the results of the immunohistochemical staining were somewhat different in this case compared to the cases of the same tumor previously described in literature. Based on that, it could be hypothesized that there was more then one cellular origin of the hemangioma in the wild boar.

Z. Lipej, B. Šoštarić, K. Hrvačić*, Ž. Mihaljević
Croatian Veterinary Institute, Zagreb, Croatia
*Ava Veterinary Clinic for small animals, Velika Gorica, Croatia

One year old Russian terrier bread bitch, previously imported from Russia was brought to veterinary clinic because of condition loss and inapetence. Despite of vigorous treatment with antibiotics and supportive therapy animal remained febrile and anorectic for ten consecutive days when was euthanized practically ante finem.
Autopsy revealed generalized malignancy.Throughout the lungs myriad of yellow-grayish nodules varying in size from almost imperceptible ones to those of several centimeters in diameter were seen.The heart was severely involved as well, and it was estimated that some 75 % of myocardium was replaced by numerous tumor masses. The liver contained large number of tumor masses of different size. Pleura, peritoneum, pancreas, kidneys, lymph nodes of practically entire body economy contained neoplastic nodules as well.
On histological examination the tumor in all involved organs was of comparable architecture and cell type, and based on characteristic histological features the diagnosis of anaplastic, small-cell type lung carcinoma was postulated.
The presented case is unusual because the original lung tumors in dogs are very rare. Further more the average age of dogs with lung malignancies is much more advanced than in observed case.